Genomic and biological insights of bacteriophages JNUWH1 and JNUWD in the arms race against bacterial resistance

Abstract

The coevolution of bacteria and bacteriophages has created a great diversity of mechanisms by which bacteria fight phage infection, and an equivalent diversity of mechanisms by which phages subvert bacterial immunity. Effective and continuous evolution by phages is necessary to deal with coevolving bacteria. In this study, to better understand the connection between phage genes and host range, we examine the isolation and genomic characterization of two bacteriophages, JNUWH1 and JNUWD, capable of infecting Escherichia coli. Sourced from factory fermentation pollutants, these phages were classified within the Siphoviridae family through TEM and comparative genomic analysis. Notably, the phages exhibited a viral burst size of 500 and 1,000 PFU/cell, with latent periods of 15 and 20 min, respectively. They displayed stability over a pH range of 5 to 10, with optimal activity at 37°C. The complete genomes of JNUWH1 and JNUWD were 44,785 bp and 43,818 bp, respectively. Phylogenetic analysis revealed their close genetic relationship to each other. Antibacterial assays demonstrated the phages' ability to inhibit E. coli growth for up to 24 h. Finally, through laboratory-driven adaptive evolution, we successfully identified strains for both JNUWH1 and JNUWD with mutations in receptors specifically targeting lipopolysaccharides (LPS) and the lptD gene. Overall, these phages hold promise as additives in fermentation products to counter E. coli, offering potential solutions in the context of evolving bacterial resistance.

Keywords: Escherichia coli; LPS; Siphoviridae; bacteriophages; genomic analysis; lptD.

Figure 1

Escherichia phage JNUWH1 and JNUWD. (A) Transmission electron micrograph of phage JNUWH1; (B) transmission electron micrograph of phage JNUWD; (C) plaques formed on the lawn of E. coli BL21.

Figure 2

Biological characteristics of phage JNUWH1 and JNUWD. (A) Thermostability and Optimal infection temperature of phage JNUWH1 and JNUWD, the line graph represents the temperature stability, and the bar graph represents the optimal infection temperature. (B) pH stability of JNUWH1 and JNUWD. The error bars indicate the standard deviation. The result was calculated based on three repeated experiments.

Figure 3

The infection curve and the one-step growth curves of JNUWH1 and JNUWD. (A) The horizontal axis represents the coincubation time of JNUWH1 at different MOIs. (B) The horizontal axis represents the coincubation time of JNUWD at different MOIs (C) One step growth curves of JNUWH1 and JNUWD in the presence of the E. coli BL21 host. The error bars indicate standard deviation. The result was calculated based on three repeated experiments.

Figure 4

(A) Genome map and functional protein identity of phage JNUWH1. (B) Genome map and functional protein identity of phage JNUWD. The arrows indicate the direction of transcription of each gene. Structurally related ORFs are marked in red, DNA-or enzyme-related ORFs are marked in green, and hypothetical proteins are marked in gray.

Figure 5

Molecular Phylogenetic analysis by Maximum Likelihood method. (A) Phylogenetic tree based on sequence of terminase large subunit. (B) Phylogenetic tree based on sequence of tail fiber protein.

Figure 6

(A) Adsorption properties of phage JNUWH1 (B) Adsorption properties of phage JNUWD. (C) The site of mutation in the lptDE of mutant M21. The error bars indicate standard deviation. ***, p < 0.001. The result was calculated based on three repeated experiments.